Metal ions contribute to fundamental biological processes, such as maintenance of protein structure, enzyme activity and signal transduction. Transport mechanisms for divalent metal ions are not well-characterized. Only recently a novel ion channel, TRPM7, was found to conduct significant amounts of these ions into cells. TRPM7 is a ubiquitously expressed protein combining a channel and a kinase domain (channel kinase). Its channel activity is regulated by intracellular magnesium (Mg2*) and nucleotriphosphates (Mg-NTP). The channel plays a key role in Mg homeostasis due to its unprecedented permeation preference for divalent ions. TRPM7 is highly conserved across species, including zebrafish, emphasizing its critical role in cell physiology. An additional protein, TRPM6, is structurally similar to TRPM7, but mainly expressed in absorptive tissues such as kidney and intestine. Dysfunctional TRPM6 activity leads to hypomagnesemia in humans. We here hypothesize that TRPM7 plays a ubiquitous role in regulating cellular magnesium homeostasis and that co-assembly with TRPM6 enhances its function in specialized absorptive tissues. We hypothesize that the alpha kinase domains of both proteins serve a dual function by regulating ion channel sensitivity to intracelLular Mg2* and Mg-ATP and by phosphorylating substrates unrelated to channel function. Hence, in Aim1 we suggest electrophysiological analyses in collaboration with Project 2 (Ryazanov) and Project 4 (Stokes) investigating the functional consequences of channel kinase heteromerization on magnesium and metal ion influx. This will be pursued in aco-expression system using HEK293 cells and in a native system from cells isolated from wild-type, heterozygous channel kinase knock-out (KO) and kinase-mute mice models. In Aim 2 we will identify structural features of the pore region of TRPM7 and TRPM6 that confer the selectivity filter for divalent ions, including magnesium. In collaboration with Project 2 (Ryazanov) a series of pore mutants will be tested for their permeation profile in both TRPM7 and TRPM6/TRPM7 heteromeric channels, leading to a delineation of the molecular basis for channel selectivity. Together with Project 3 (Scharenberg) we will determine in Aim3 whether the biophysical and functional properties of TRPM7 and TRPM6/TRPM7 are conserved across species. Based on our current knowledge of TRPM7 physiology we will focus on the biophysical characterization of zebrafish channel kinase. Further differential analyses will identify regions of channel kinases responsible for specific physiological functions. Detailed understanding of the functional consequences of TRPM6/TRPM7 interaction and role of their kinase domains will open new possibilities to influence magnesium and metal ion absorption in health and disease.